IMMUNOGENIC RESPIRATORY SYNCYTIAL VIRUS GLYCOPROTEIN-CONTAINING VLPs AND RELATED COMPOSITIONS, CONSTRUCTS, AND THERAPEUTIC METHODS

ABSTRACT

The invention provides immunotherapeutic and prophylactic bacteriophage viral-like particle (VLPs) which are useful in the treatment and prevention of Respiratory Syncytial Virus (RSV) infections and related disorders, including bronchiolitis and viral pneumonia. Related compositions (e.g. vaccines), nucleic acid constructs, and therapeutic methods are also provided. VLPs and related compositions of the invention induce high titer antibody responses against RSV. VLPs, VLP-containing compositions, and therapeutic methods of the invention induce an immunogenic response against RSV infection, confer immunity against RSV infection, protect against RSV infection, and reduce the likelihood of infection by RSV infection.

RELATED APPLICATIONS AND GOVERNMENT INTEREST

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 61/382,704, of identical title, filed Sep. 14,2010, the entire contents of which is incorporated by reference herein.

This invention was made with government support under Grant No. GM042901awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

In one aspect, the invention provides immunogenic Respiratory SyncytialVirus (RSV) glycoprotein-containing viral-like particles (VLPs). Incertain aspects, the invention provides immunogenic RSV G and Fglycoprotein-containing VLPs. Related compositions (e.g. vaccines),nucleic acid constructs, and therapeutic methods are also provided.

In one aspect, the invention relates generally to virus-like particlesand, more specifically, to a platform for peptide display based on VLPsof the RNA bacteriophage PP7.

In certain aspects, the VLPs are comprised of a coat polypeptide of thebacteriophages PP7 or MS2, wherein the coat protein is modified byinsertion of peptide antigens derived from a RSV glycoprotein (e.g. aRSV G or F glycoprotein), the recombinant coat protein is expressed toproduce a VLP, and wherein the RSV G or F glycoprotein peptide isdisplayed on the surface of the VLP.

Immunogenic VLPs and related compositions of the invention induce hightiter antibody responses against a RSV glycoprotein (e.g. a RSV G or Fglycoprotein) and may serve as a prophylactic or therapeutic vaccine forRSV infection.

BACKGROUND OF THE INVENTION

Respiratory Syncytial Virus (RSV) is a human pathogen that is thepredominant cause of acute lower respiratory tract infection in children[reviewed by (27)]. In the United States, nearly all children areinfected with RSV by the age of three. Symptoms range from severepneumonia and bronchiolitis to milder infections. Because of itsprevalence, RSV is a major cause of serious respiratory illnessrequiring hospitalization in children. Significant morbidity andmortality are also associated with RSV infection of the elderly.

The viral genome encodes two major structural glycoproteins that areexposed on the surface of RSV virions, G and F. G is believed to play arole in viral attachment to a cellular receptor. F promotes fusion ofthe viral and cell membranes, allowing entry of the viral genome intothe target cell. Antibodies against either G or F can neutralize RSVinfectivity (18). The G protein is highly genetically diverse amongstRSV strains, thus anti-G neutralizing antibodies are only neutralizingagainst viruses of one of the two antigenic groups of RSV (either A orB). In contrast, F is highly conserved and anti-F neutralizingantibodies can protect animal models from infection by both group-A andgroup-B viruses (26, 31).

There is no current vaccine for RSV infection. (In the 1960s aformalin-inactivated RSV vaccine candidate actually increased diseaseseverity in vaccinated children.) Moreover, there are no usefultherapeutics for treating RSV infection. However, Palivizumab, alsoknown by its trade name Synagis, a monoclonal antibody that targets aneutralizing epitope in the F glycoprotein, has been usedprophylactically to prevent RSV infection. Although Synagis iseffective, it is very expensive and only is cost-effective for use ininfants that are at extremely high risk for developing severe RSVinfection. Because of its expense, it is only used in developedcountries.

Virus-Like Particle (VLP) Based Vaccines.

Virus-like particles (VLPs) make excellent vaccines. They arenon-infectious, often easier to produce than actual viruses, and,because the regularity of their capsid structure presents viral epitopesas dense, highly repetitive arrays that strongly stimulate B cells, theyare highly immunogenic. VLPs are comprised of one or more proteinsarranged geometrically into dense, repetitive arrays. These structuresare largely unique to microbial antigens, and the mammalian immunesystem has apparently evolved to respond vigorously to this arrangementof antigens. B cells specifically recognize and respond strongly to theordered array of densely spaced repetitive elements characteristic ofvirus surfaces (1, 16). Highly repetitive antigens provokeoligomerization of the membrane-associated immunoglobulin (Ig) moleculesthat constitute the B cell receptor (BCR) (2). There is evidence thatthe Ig crosslinking mediated by multivalent antigens leads to theformation of highly stable BCR-signaling microdomains that areassociated with increased signaling to the B cell (34). This signalingstimulates B cell proliferation, migration, and upregulation of bothmajor histocompatibility complex (MHC) class II and the co-stimulatorymolecules that permit subsequent interactions with T helper cells thatare required to trigger IgG secretion, affinity maturation, and thegeneration of long-lived memory B cells (8). Consequently, multivalentantigens such as VLPs can activate B cells at much lower concentrationsthan monomeric antigens (3, 13, 14, 25). Hence, VLPs are innatelyimmunogenic: they induce high titer and long lasting antibody responsesat low doses, often without requiring adjuvants (19, 36).

VLPs as Flexible Platforms for Vaccine Development.

VLPs can be used as the basis for vaccines targeting the virus fromwhich they were derived (the Hepatitis B virus vaccine and HPV vaccineare two clinically approved VLP vaccines, other VLP vaccines are inclinical trials). However, they also can be used as platforms to displaypractically any epitope in a highly immunogenic, multivalent format.Heterologous antigens displayed at high density on the surface of VLPsexhibit the same high immunogenicity as unmodified VLPs. VLPs derivedfrom a variety of different viruses have been exploited in this mannerto induce antibody responses against heterologous targets that arepoorly immunogenic in their native contexts. Although the VLP platformstrategy has typically been applied to target antigens derived frompathogens, VLP-display can effectively induce antibody responses againstpractically any antigen. One example is the vaccine for nicotineaddiction (designed to assist smokers who are trying to quit) developedby a biotechnology company, Cytos Biotechnology. This vaccine consistsof nicotine, conjugated at high copy number to the surface of VLPsderived from a bacteriophage. In phase II clinical trials, VLPsdisplaying nicotine were well-tolerated and induced strongnicotine-specific IgG responses in 100% of immunized subjects (12). Evenself-antigens, which are normally subject to the mechanisms of B celltolerance, are immunogenic when displayed at high density on the surfaceof VLPs. Vaccines have been developed against self-molecules involved inseveral different diseases, including amyloid-beta (Alzheimers (11,21)), TNF-α (arthritis (9)), CCR5 (HIV infection (7, 10)), gastrin(cancer, unpublished data), IgE (allergy, unpublished data), and others.VLP-based vaccines developed by pharmaceutical companies targetingamyloid-beta and angiotensin II (hypertension) are currently beingevaluated in clinical trials; positive results from the trial of vaccinetargeting angiotensin II (as a vaccine for hypertension) were reportedin the spring of 2008 (35).

Objects of the Invention

It is an object of the invention to provide a virus-like particle (VLP)virus-like particle comprising a bacteriophage single chain coatpolypeptide dimer, preferably based upon a MS2 or PP7 bacteriophage anda RSV peptide (preferably an epitope within RSV glycoprotein G or F,wherein the RSV peptide is displayed on the virus-like particle and saidVLP encapsidates bacteriophage mRNA, such that the composition isimmunotherapeutic and prophylactic for RSV infections and/or RSV-induceddisorders and/or secondary disease states and conditions.

It is another object of the invention to provide nucleic acid constructswhich express a VLP comprising a bacteriophage single chain coatpolypeptide dimer, preferably based upon a MS2 or PP7 bacteriophage andan epitope within RSV glycoprotein G or F, wherein the RSV peptide isdisplayed on the viral-like particle and wherein said VLP encapsidatesbacteriophage mRNA.

It is another object of the invention to provide a method of instillingimmunogenicity or prophylaxis to a RSV infection and/or a RSV relateddisorder in a patient at risk for such an infection or disorder.

Providing an immunogenic response to a RSV peptide in a subjectrepresents an additional object of the invention.

Providing a vaccine against RSV infection and/or an RSV-related disorderwith low toxicity and minimal side effects represents a further objectof the present invention.

SUMMARY OF THE INVENTION

The need exists for a cost-effective, widely-applicable RSV vaccine thatcan be used in the inoculation and treatment of a broad-range ofpatients, including infants who are at a high risk for developing severeRSV infection.

Previously we described the use of virus-like particles (VLPs) of twoRNA bacteriophages, MS2 and PP7, for peptide display (6, 30). MS2 andPP7 coat protein single-chain dimers are highly tolerant of peptideinsertions and produce correctly assembled VLPs displaying the peptideinsertion on the surface of VLP in a highly dense, repetitive array.These VLPs are highly immunogenic and confer this high immunogenicity toheterologous peptides displayed on their surfaces. Here we describe VLPsdisplaying peptide antigens derived from Respiratory Syncytial Virus(RSV) glycoproteins such as RSV glycoproteins G and F. Such recombinantVLPs serve as an immunogenic material and prophylactic vaccine toprevent infection by RSV.

The invention provides immunotherapeutic and prophylactic bacteriophageviral-like particle (VLPs) which are useful in the treatment andprevention of RSV and related disorders. Related compositions (e.g.vaccines), nucleic acid constructs, and therapeutic methods are alsoprovided. VLPs and related compositions of the invention induce hightiter antibody responses against RSV. VLPs, VLP-containing compositions,and therapeutic methods of the invention induce an immunogenic responseagainst RSV neutralizing epitopes.

Because antibodies that are specific for highly conserved epitopeswithin RSV glycoproteins G and F are able to neutralize infection by abroad range of RSV types, RSV glycoproteins G and F-targeting VLPs andrelated compositions (e.g. vaccines) of the invention provide a morecomprehensive protection against infection by multiple RSV types.

In one aspect, the invention provides a VLP comprising a bacteriophagesingle chain coat polypeptide dimer and a RSV peptide (e.g. a RSV G or Fpeptide, preferably F peptide of about 3 to about 30, more preferablyabout 5 to about 20, about 5 to about 15, about 5 to about 10 amino acidunits in length), wherein the RSV peptide is displayed on the VLP, andwherein the VLP produces an immunogenic response in a subject and/or isimmuno-prophylactic for RSV-induced disorders.

Certain aspects of the invention reflect that the single-chain dimer ofMS2 and PP7 coat protein can tolerate the insertion of a wide variety ofpeptides, including peptides derived from different strains of RSV, canself-assemble into VLPs, and is highly immunogenic.

In another aspect, the invention provides a composition comprising a VLPcomprising a bacteriophage single chain coat polypeptide dimer and a RSVpeptide (e.g. a RSV G or F peptide, preferably F peptide of 3 to 30,more preferably 5 to 20 amino acid units in length), wherein the RSVpeptide is displayed on the VLP, and wherein the composition isimmunotherapeutic and prophylactic for RSV-induced disorders.

In certain aspects, the invention provides a VLP, or a compositioncomprising a VLP, wherein the VLP is made by transforming a prokaryotewith a nucleic acid construct comprising:

-   -   (a) a bacterial or bacteriophage promoter;    -   (b) a coding sequence of a bacteriophage single chain coat        polypeptide dimer which is operably associated with the promoter        and which is modified to contain a nucleotide sequence encoding        a RSV peptide (e.g. RSV G or F peptide, preferably F peptide of        3 to 30, more preferably 5 to 20 amino acid units in length);    -   (c) an antibiotic repressor which is operably associated with        the promoter; and    -   (d) a replication origin for replication in a prokaryotic cell,        wherein the composition is immunotherapeutic and prophylactic        for RSV-induced disorders.

In certain aspects, VLPs and VLP-containing compositions (e.g. vaccines)of the invention are comprised of VLPs comprising RSV peptides fromdifferent RSV types. In other aspects, VLPs and VLP-containingcompositions of the invention comprise hybrid VLPs that display multipleRSV sequences.

In certain aspects, the invention provides a VLP, or a compositioncomprising a VLP, wherein the VLP is made by transforming a prokaryotewith a nucleic acid construct comprising either:

-   -   (1) (a) a bacterial or bacteriophage promoter which is operably        associated with a coding sequence of a bacteriophage (preferably        PP7) single chain coat polypeptide dimer, wherein the coat        polypeptide dimer coding sequence is modified to: (i) define a        first restriction site which is located in the downstream        portion of the coat polypeptide dimer coding sequence and which        is either positioned 5′ to, or located within, the sequence        which defines the coat polypeptide dimer AB loop, and (ii)        contain a nucleotide sequence encoding a RSV peptide (e.g. a RSV        G or F peptide, preferably a F peptide of 3 to 30, more        preferably 5 to 20 amino acid units in length);    -   (b) a second restriction site positioned 3′ to the coat        polypeptide dimer coding sequence;    -   (c) an antibiotic repressor which is operably associated with        the promoter; and    -   (d) a replication origin for replication in a prokaryotic cell;        or    -   (2) (a) a bacterial or bacteriophage promoter which is operably        associated with a coding sequence of a bacteriophage (preferably        PP7), single chain coat polypeptide dimer, wherein the coat        polypeptide dimer coding sequence is modified to: (i) define a        first restriction site which is located in the downstream        portion of the coat polypeptide dimer coding sequence and which        is either positioned 5′ to, or located within, the sequence        which defines the coat polypeptide dimer AB loop, and (ii)        contain a nucleotide sequence encoding a RSV peptide (e.g. a RSV        G or F peptide, preferably a F peptide of 3 to 30, more        preferably 5 to 20 amino acid units in length);    -   (b) a second restriction site positioned 3′ to the coat        polypeptide dimer coding sequence;    -   (c) a PCR primer positioned 3′ to the second restriction site;    -   (d) an antibiotic repressor which is operably associated with        the promoter; and    -   (e) a replication origin for replication in a prokaryotic cell;        or    -   (3) (a) a bacterial or bacteriophage promoter which is operably        associated with a coding sequence of a bacteriophage (preferably        MS2) single chain coat polypeptide dimer, wherein the coat        polypeptide dimer coding sequence is modified to (i) define a        codon sequence positioned 5′ to that portion of the sequence        which defines the coat polypeptide dimer AB loop, and (ii)        contain a nucleotide sequence encoding a RSV peptide (e.g. a RSV        G or F peptide, preferably a F peptide of 3 to 30, more        preferably 5 to 20 amino acid units in length);    -   (b) a restriction site positioned 3′ to the coat polypeptide        dimer coding sequence;    -   (c) a PCR primer positioned 3′ to the second restriction site;    -   (d) a repressor to resistance to a first antibiotic, wherein the        repressor is operably associated with the promoter;    -   (e) a helper phage gene modified to contain a gene conferring        resistance to a second antibiotic, and    -   (f) a replication origin for replication in a prokaryotic cell.

In certain aspects, the invention provides a method of generating animmunogenic response to a RSV peptide, the method comprisingadministering to a subject an effective amount of a RSVpeptide-containing VLP as otherwise described herein, optionally incombination with a pharmaceutically acceptable carrier, additive orexcipient. In certain aspects, the invention provides a method ofinoculating a subject at risk of developing a RSV infection or aRSV-related disorder, the method comprising administering to the subjectone or more doses of a composition comprising a RSV peptide-containing(e.g. RSV G or F peptide-containing, preferably F peptide-containing of3 to 30, more preferably 5 to 20 amino acid units in length) VLP asdescribed herein. In other aspects, the invention provides a method oftreating a subject who is at risk of developing a RSV-related disorderand who has undergone RSV seroconversion, the method comprisingadministering to the subject one or more doses of a compositioncomprising a RSV peptide containing VLP as described herein. In stillother aspects, the invention provides a method of treating a subject whohas developed a RSV-related disorder, the method comprisingadministering to the subject one or more doses of a compositioncomprising a RSV-containing VLP as described herein.

Previously, we described the use of VLPs of the RNA bacteriophage MS2for peptide display. By genetically fusing two copies of the MS2 coatprotein, we created a single-chain dimer with increased thermodynamicstability and vastly improved tolerance of insertions in its AB-loop(30). The MS2 coat protein dimer was widely tolerant of geneticinsertion of defined peptide sequences as well as random peptideinsertions. Recombinant MS2 VLPs elicited high titer IgG antibodiesagainst the inserted sequences. Moreover, MS2 coat protein single-chaindimers produced correctly assembled VLPs that specifically encapsidatedthe mRNA encoding their synthesis, raising the possibility that theycould be used in affinity selections protocols analogous to filamentousphage display.

Since MS2 is only one member of a large family of bacteriophages whoseindividual members share similar molecular biology, we suspected that,following similar design principles, other phage VLPs could be adaptedto this same purpose. For example, we include hereinafter our previousdescription in U.S. Provisional Patent Application Ser. Nos. 61/302,836,filed Feb. 9, 2010, 61/334,826, filed May 14, 2010 and PCT applicationno. PCT/US2011/24030, filed 8 Feb. 2011 (the complete contents of whichare hereby incorporated by reference) of the engineering of VLPs of PP7,a bacteriophage of Pseudomonas aeruginosa, for the purposes of peptidedisplay.

PP7 VLPs offer several potential advantages and improvements over theMS2 VLP. First, the particles are dramatically more stablethermodynamically, because of the presence of stabilizing inter-subunitdisulfide bonds. For many practical applications, including vaccines,increased stability is a desirable trait. Second, PP7 VLPs are notcross-reactive immunologically with those of MS2. This could beimportant in applications where serial administration of VLPs may benecessary. Third, we anticipated that the correct folding and assemblyof the PP7 VLP might be more resistant to the destabilizing effects ofpeptide insertion, or that it might at least show tolerance of somepeptides not tolerated in MS2 VLPs.

The single-chain dimer of PP7 coat protein can tolerate the insertion ofa wide variety of peptides, is highly immunogenic, and packages the RNAthat directs its synthesis. Moreover, we have shown in United StatesProvisional Patent Application Serial No. U.S. Provisional PatentApplication Ser. Nos. 61/302,836, filed Feb. 9, 2010, 61/334,826, filedMay 14, 2010 and PCT application no. PCT/US2011/24030, filed 8 Feb. 2011(the complete contents of which are hereby incorporated by reference)that an in vivo challenge model that a PP7 VLP displaying a broadlycross-type neutralizing epitope from the HPV minor capsid protein L2induces antibodies that protect against homologous and heterologous HPVinfection.

Thus, we describe the use of recombinant VLPs derived RNA bacteriophagesto induce high titer antibody responses against RSV G or F peptide thatprotect against multiple diverse RSV types.

These and other aspects of the invention are described further in theDetailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A. The nucleotide and amino acid sequence of target regions inthe RSV F and G glycoproteins. B. The N-terminal sequence the downstreamcopy of coat protein encoded by pDSP62 (MS2) or p2P7K32 (PP7). C. A listof the forward primers used to clone the listed sequences into MS2 coat(shown 5′ to 3′). The SalI restriction site is shown in italics and thepeptide insertion is shown in bold text. A similar strategy was used toclone these sequences into the PP7 expression vector. Sequences wereinserted into PP7 at amino acid 11, resulting in a duplication of thisresidue [as described in (6)].

FIG. 2. Anti-RSV antibody responses induced by recombinant MS2 VLPs.Groups of three mice were immunized three times (at weeks 0, 2, and 5)with 10 μg of VLPs plus incomplete Freund's adjuvant. Two weeks afterthe final immunization mice were bled and sera from individual mice weretested for reactivity with a peptide representing the “Synagis” epitope(Leu-Thr-Asn-Ser-Glu-Leu-Leu-Ser-Leu-Ile-Asn-Asp-Met-Pro-Ile-Thr-Asn-Asp-Gln-Lys-Lys-Leu-Met-Ser-Asn-Asn-Val)(panel A), or pooled sera from each group were tested for reactivitywith recombinant F antigen (purchased from Sino Biological Inc.; panelB), or with UV-inactivated RSV virions (purchased from FitzgeraldIndustries; panels C and D) by ELISA. The results above show the IgGantibody titer for individual mice (panel A) or ELISA values(represented as optical density at 405 nm; OD405, panels B, C, and D)for pooled sera from each group of three mice at various serumdilutions. Error bars indicate the standard error of the mean (SEM).

FIG. 3 depicts the pDSP1 plasmid and a technique for inserting a nucleicacid sequence encoding a heterologous peptide into that plasmid.

FIG. 4 (N12-150) contains the nucleic acid sequence for the pDSP1plasmid (SEQ ID NO: 1).

FIG. 5 shows the nucleotide sequence the plasmid pDSP62 containing the Fantigen peptide S10.5 inserted into the AB-loop of one copy of thesingle-chain dimer (SEQ ID NO: 2).

FIG. 6 depicts the p2P7K32 plasmid.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984,“Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal and preferablya human, to whom treatment, including prophylactic treatment(prophylaxis), with the immunogenic compositions and/or vaccinesaccording to the present invention is provided to inoculate and/orgenerate an immunogenic response. For treatment of those infections,conditions or disease states which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal. In most instances, the patient or subject of the presentinvention is a human patient of either or both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe a number of VLP's or an amount of a VLP-containing compositionwhich, in context, is used to produce or effect an intended result,whether that result relates to the prophylaxis and/or therapy of a RSVinfection or RSV-induced or RSV-related disorder (or secondary diseasestate or condition) or as otherwise described herein. The term effectivesubsumes all other effective amount or effective concentration terms(including the term “therapeutically effective”) which are otherwisedescribed or used in the present application.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded DNA and RNA. Apolynucleotide may include nucleotide sequences having differentfunctions, such as coding regions, and non-coding regions such asregulatory sequences (e.g., promoters or transcriptional terminators). Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers (eg., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional is retained by the polypeptide. NH₂ refers to the free aminogroup present at the amino terminus of a polypeptide. COOH refers to thefree carboxy group present at the carboxy terminus of a polypeptide.

The term “single-chain dimer” refers to a normally dimeric protein whosetwo subunits have been genetically (chemically, through covalent bonds)fused into a single polypeptide chain. Specifically, in the presentinvention single-chain dimer versions of PP7 coat proteins wereconstructed. Each of these proteins is naturally a dimer of identicalpolypeptide chains. In the PP7 coat protein dimers the N-terminus of onesubunit lies in close physical proximity to the C-terminus of thecompanion subunit. Single-chain coat protein dimers were produced usingrecombinant DNA methods by duplicating the DNA coding sequence of thecoat proteins and then fusing them to one another in tail to headfashion. The result is a single polypeptide chain in which the coatprotein amino acid appears twice, with the C-terminus of the upstreamcopy covalently fused to the N-terminus of the downstream copy. Normally(wild-type) the two subunits are associated only through noncovalentinteractions between the two chains. In the single-chain dimer thesenoncovalent interactions are maintained, but the two subunits haveadditionally been covalently tethered to one another. This greatlystabilizes the folded structure of the protein and confers to it itshigh tolerance of peptide insertions as described above.

VLPs according to the present invention (especially those based upon PP7and MS2 coat polypeptide dimers), because of their use of single-chaindimers as described above, exhibit a number of advantages overtraditional approaches for providing vaccines, including vaccines basedupon prior art viral particles. For examples, VLPs according to thepresent invention exhibit exceptional stability, ease of manufacture,higher yields during manufacturing and they are regular in appearancewith greater consistency, resulting in a more reliable immunogenicresponse with lower toxicity and fewer side effects.

This application makes reference to coat protein's “AB-loop”. The RNAphage coat proteins possess a conserved tertiary structure. The PP7 coatproteins, for example, possess a structure wherein each of thepolypeptide chains is folded into of a number of β-strands. Theβ-strands A and B form a hairpin with a three-amino acid loop connectingthe two strands at the top of the hairpin, where it is exposed on thesurface of the VLP. As evidenced in the present application, peptidesinserted into the AB-loop are exposed on the surface of the VLP and arestrongly immunogenic.

The term “valency” is used to describe the density of a RSV peptidedisplay on VLPs according to the present invention. Valency in thepresent invention may range from low valency to high valency, about lessthan 1 to more than about 180, preferably about 90 to 180. Immunogeniccompositions according to the present invention comprise VLPs which arepreferably high valency and comprise VLPs which display at least 50-60up to about 180 or more RSV peptides.

The term “coding sequence” is defined herein as a portion of a nucleicacid sequence which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′-end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′-end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

A “heterologous” region of a recombinant cell is an identifiable segmentof nucleic acid within a larger nucleic acid molecule that is not foundin association with the larger molecule in nature.

An “origin of replication”, used within context, normally refers tothose DNA sequences that participate in DNA synthesis by specifying aDNA replication initiation region. In the presence of needed factors(DNA polymerases, and the like) an origin of replication causes orfacilitates DNA associated with it to be replicated. By way of anon-limiting example, the ColE1 replication origin endows many commonlyused plasmid cloning vectors with the capacity to replicateindependently of the bacterial chromosome. Another example is the p15Areplication origin. The presence on a plasmid of an additional origin ofreplication from phage M13 confers the additional ability to replicateusing that origin when E. coli cells are infected with a so-calledhelper phage (e.g. M13CM1) which provides necessary protein factors. M13replicates intracellularly as double-stranded circular DNA, but alsoproduces a single-stranded circular form, which it packages within thephage particle. These particles provide a convenient source ofsingle-stranded circular DNA for plasmids.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence includes the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter will be found DNA sequences responsiblefor the binding of RNA polymerase and any of the associated factorsnecessary for transcription initiation. In bacteria promoters normallyconsist of −35 and −10 consensus sequences and a more or less specifictranscription initiation site. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Bacterial expressionvectors (usually plasmids or phages) typically utilize promoters derivedfrom natural sources, including those derived from the E. coli Lactose,Arabinose, Tryptophan, and ProB operons, as well as others frombacteriophage sources. Examples include promoters from bacteriophageslambda, T7, T3 and SP6.

In bacteria, transcription normally terminates at specific transcriptiontermination sequences, which typically are categorized as rho-dependentand rho-independent (or intrinsic) terminators, depending on whetherthey require the action of the bacterial rho-factor for their activity.These terminators specify the sites at which RNA polymerase is caused tostop its transcription activity, and thus they largely define the3′-ends of the RNAs, although sometimes subsequent action ofribonucleases further trims the RNA.

An “antibiotic resistance gene” refers to a gene that encodes a proteinthat renders a bacterium resistant to a given antibiotic. For example,the kanamycin resistance gene directs the synthesis of aphosphotransferase that modifies and inactivates the drug. The presenceon plasmids of a kanamycin resistance gene provides a mechanism toselect for the presence of the plasmid within transformed bacteria.Similarly, the chloramphenicol resistance gene allows bacteria to growin the presence of the drug by producing an acetyltransferase enzymethat inactivates the antibiotic through acetylation.

The term “PCR” refers to the polymerase chain reaction, a technique usedfor the amplification of specific DNA sequences in vitro. The term “PCRprimer” refers to DNA sequences (usually synthetic oligonucleotides)able to anneal to a target DNA, thus allowing a DNA polymerase (e.g. TaqDNA polymerase) to initiate DNA synthesis. Pairs of PCR primers are usedin the polymerase chain reaction to initiate DNA synthesis on each ofthe two strands of a DNA and to thus amplify the DNA segment between twoprimers. Representative PCR primers which used in the present inventionare those which are presented in FIG. 1 hereof. Additional PCR primersmay be obtained for the various RSV peptides which are presented herein.

Examples of primers used for PCR are given in FIG. 1 as described aboveand the following.

E3.2: 5′ CGG GCT TTG TTA GCA GCC GG 3′—(SEQ ID No. 3) may serve as the3′ (reverse)-primer in PCR reactions to amplify coat protein.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional controlsequences are DNA regulatory sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide for theexpression of a coding sequence in a host cell, Translational controlsequences determine the efficiency of translation of a messenger RNA,usually by controlling the efficiency of ribosome binding andtranslation initiation. For example, as discussed elsewhere in thisapplication, the coat proteins of the RNA phages are well-knowntranslational repressors of the phage replicase. As coat proteinaccumulates to a sufficiently high concentration in the infected cell,it binds to an RNA hairpin that contains the translation initiationregion (Shine-Dalgarno and initiator AUG) of the phage's replicase gene.This prevents ribosome binding and shuts off replicase synthesis at atime in the viral life cycle where the transition from replication tovirus assembly occurs.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which aredegenerate to the nucleic acids disclosed herein. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

It should be appreciated that also within the scope of the presentinvention are nucleic acid sequences encoding the polypeptide(s) of thepresent invention, which code for a polypeptide having the same aminoacid sequence as the sequences disclosed herein, but which aredegenerate to the nucleic acids disclosed herein. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid.

As used herein, “epitope” refers to an antigenic determinant of apolypeptide, in this case a RSV peptide. An epitope could comprise 3amino acids in a spatial conformation which is unique to the epitope.Generally an epitope consists of at least 5 such amino acids, and moreusually, consists of at least about 8-10 up to about 20 or more suchamino acids. Methods of determining the spatial conformation of aminoacids are known in the art, and include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance.

As used herein, a “mimotope” is a peptide that mimics an authenticantigenic epitope, which epitope may include both peptide andcarbohydrate epitopes.

As used herein, the term “coat protein(s)” refers to the protein(s) of abacteriophage or a RNA-phage capable of being incorporated within thecapsid assembly of the bacteriophage or the RNA-phage.

As used herein, a “coat polypeptide” as defined herein is a polypeptidefragment of the coat protein that possesses coat protein function andadditionally encompasses the full length coat protein as well orsingle-chain variants thereof.

As used herein, the term “immune response” refers to a humoral immuneresponse and/or cellular immune response leading to the activation orproliferation of B- and/or T-lymphocytes and/or antigen presentingcells. In some instances, however, the immune responses may be of lowintensity and become detectable only when using at least one substancein accordance with the invention. “Immunogenic” refers to an agent usedto stimulate the immune system of a living organism, so that one or morefunctions of the immune system are increased and directed towards theimmunogenic agent. An “immunogenic polypeptide” is a polypeptide thatelicits a cellular and/or humoral immune response, whether alone orlinked to a carrier in the presence or absence of an adjuvant.Preferably, antigen presenting cell may be activated.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal and provides ameasure of protection (protective effect) against a disease state orcondition for which the vaccine is administered. The term “prevention”is used in context synonymously with the term “reducing the likelihoodof” or “inhibiting” wherein the measure of prevention (of a diseasestate or condition) is one of degree of effect. Vaccines according tothe present invention instill immunogenicity against a RSV peptide andmay also instill immunity in a patient or subject against a diseasestate or condition (RSV or a related condition or disease state).

As used herein, the term “virus-like particle of a bacteriophage” refersto a virus-like particle (VLP) resembling the structure of abacteriophage, being non replicative and noninfectious, and lacking atleast the gene or genes encoding for the replication machinery of thebacteriophage, and typically also lacking the gene or genes encoding theprotein or proteins responsible for viral attachment to or entry intothe host. This definition should, however, also be seen to encompassvirus-like particles of bacteriophages, in which the aforementioned geneor genes are still present but inactive, and, therefore, also leading tonon-replicative and noninfectious virus-like particles of abacteriophage.

VLP of RNA bacteriophage coat protein: The capsid structure formed fromthe self-assembly of one or more subunits of RNA bacteriophage coatprotein and optionally containing host RNA is referred to as a “VLP ofRNA bacteriophage coat protein”. In a particular embodiment, the capsidstructure is formed from the self assembly of 90-180 subunits.

A nucleic acid molecule is “operatively linked” to, or “operablyassociated with”, an expression control sequence when the expressioncontrol sequence controls and regulates the transcription andtranslation of nucleic acid sequence. The term “operatively linked”includes having an appropriate start signal (e.g., ATG) in front of thenucleic acid sequence to be expressed and maintaining the correctreading frame to permit expression of the nucleic acid sequence underthe control of the expression control sequence and production of thedesired product encoded by the nucleic acid sequence. If a gene that onedesires to insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

RSV-Induced Disorders, Immunogenicity, and Prophylactic Efficacy

Respiratory Syncytial Virus (RSV) is a human pathogen that is thepredominant cause of acute lower respiratory tract infection in children[reviewed by (27)]. In the United States, nearly all children areinfected with RSV by the age of three. Symptoms range from severepneumonia and bronchiolitis to milder infections. Because of itsprevalence, RSV is a major cause of serious respiratory illnessrequiring hospitalization in children. There are more than 26 strainswithin the two types/subgroups A and B of RSV. Unless otherwiseindicated, the term RSV refers to the various strains of the virus.Significant morbidity and mortality are also associated with RSVinfection of the elderly. The term “RSV-induced disorder” refers to adisease state, condition or symptomology which occurs in a patientsecondary to or, as a consequence of RSV infection, includingbronchiolitis and/or viral pneumonia and other symptomology associatedwith viral respiratory tract infection (wheezing, cough, hightemperature, etc.).

RSV Glycoproteins, Peptides, and Peptide Sequences

“RSV” as used herein includes RSV Types A and B. “RSV glycoproteinpeptide sequences” include RSV F and G glycoprotein peptide sequences,as well as all other RSV glycoprotein peptide sequences, e.g. asidentified in (17).

Preferred RSV glycoprotein peptide sequences include epitopic peptidesequences comprising from 3 to 30, preferably 5 to 20 amino acid RSV For G glycoprotein sequences, preferably from 5 to about 8-10 amino acidsof RSV F or G glycoprotein sequences, preferably F glycoproteinsequences. Exemplary RSV epitopic peptide sequences which are preferablycontained in VLP particles according to the present invention include,for example, contiguous peptide sequences of the F Antigen amino acidsequence presented hereinbelow ranging from 3 to 30, 5 to 20, 5 to 15, 5to 12, 8 to 12, preferably 8-10 amino acid units.

F Antigen amino acid sequence (SEQ ID NO: 4): 1melpilkana ittilaavtf cfassqnite efyqstcsav skgylsalrt gwytsvitie 61lsnikenkcn gtdakvklmk qeldkyknav telqllmqst paannrarre lprfmnytln 121ntkktnvtls kkrkrrflgf llgvgsaias giavskvlhl egevnkiksa llstnkavvs 181lsngvsvlts kvldlknyid kqllpivnkr scrisnietv iefqhknnrl leitrefsvn 241agvttpvsty mltnsellsl indmpitndq kklmsnnvqi vrqqsysims iikeevlayv 301vqlplygvid tpcwklhtsp lcttntkegs nicltrtdrg wycdnagsvs ffpqaetckv 361qsnrvfcdtm nsltlpsevn lcnvdifnpk ydckimtskt dvsssvitsl gaivscygkt 421kctasnknrg iiktfsngcd yvsnkgvdtv svgntlyyvn kqegkslyvk gepiinfydp 481lvfpsdefda sisqvnekin qslafirksd ellhnvnagk sttnimitti iieiivills 541liavglllyc karstpvtls kdqlsginni afsn

Epitopic peptides from the F antigen of RSV are preferred for use in thepresent invention. Particularly preferred epitopic amino acid sequencesmay be readily obtained from antigenic site II, antigenic site IV orantigenic site I of the F antigen of RSV.

Contiguous peptide sequences (e.g., about 5-20, 5-15, 5-10, 8-10contiguous amino acids) from the following sites of the F antigen, areparticularly preferred:

-   -   1. The so-called antigenic site II (amino acids 252-278) (e.g.,        3-28, 5-20, 5-15, 5-10, 6-10, 7-10, 8-10 contiguous amino acids        of amino acids 252-278 of the antigenic site II):

(SEQ ID NO: 5) LTNSELLSL INDMPITNDQ KKLMSNNV

-   -   2. Antigenic site IV (encompassing amino acids 429-437) (e.g.        3-20, 4-20, 5-20, 5-15, 5-10, 6-10, 7-10, 8-10 contiguous amino        acids of amino acids 429-437 of the antigenic site IV):

(SEQ ID NO: 6) KCTASNKNRGIIKTFSNGCD

-   -   3. Antigenic site I (encompassing amino acid 389) (e.g. 3-20,        4-20, 5-20, 5-15, 5-10, 6-10, 7-10, 8-10 contiguous amino acids        of antigenic site I):

(SEQ ID NO: 7) LCNVDIFNPKYDCKIMTSKT

Other preferred epitopic peptide sequences can be readily provided fromthe list of sequences presented above and include the following:

Any 5-20 contiguous amino acids from antigenic site II (amino acids252-278), presented above;

Any 5-15 contiguous amino acids from antigenic site II,

Any 5-12 contiguous amino acids from antigenic site II,

Any 5-10 contiguous amino acids from antigenic site II,

Any 6-11 contiguous amino acids from antigenic site II,

Any 6-10 contiguous amino acids from antigenic site II,

Any 7-10 contiguous amino acids from antigenic site II,

Any 8-10 contiguous amino acids from antigenic site II,

Any 6 contiguous amino acids from antigenic site II,

Any 7 contiguous amino acids from antigenic site II,

Any 8 contiguous amino acids from antigenic site II,

Any 9 contiguous amino acids from antigenic site II,

Any 10 contiguous amino acids from antigenic site II,

Any 11 contiguous amino acids from antigenic site II,

Any 12 contiguous amino acids from antigenic site II.

Any 5-20 contiguous amino acids from antigenic site IV (whichencompasses amino acids 429-437), presented above;

Amino acids 429-437 of antigenic site IV;

Any 5 contiguous amino acids of amino acids 429-437 of antigenic siteIV;

Any 6 contiguous amino acids of amino acids 429-437 of antigenic siteIV;

Any 7 contiguous amino acids of amino acids 429-437 of antigenic siteIV;

Either of the 8 contiguous amino acids of amino acids 429-437 ofantigenic site IV;

Any 5-15 contiguous amino acids from antigenic site IV,

Any 5-12 contiguous amino acids from antigenic site IV,

Any 5-10 contiguous amino acids from antigenic site IV,

Any 6-11 contiguous amino acids from antigenic site IV,

Any 6-10 contiguous amino acids from antigenic site IV,

Any 7-10 contiguous amino acids from antigenic site IV,

Any 8-10 contiguous amino acids from antigenic site IV,

Any 6 contiguous amino acids from antigenic site IV,

Any 7 contiguous amino acids from antigenic site IV,

Any 8 contiguous amino acids from antigenic site IV,

Any 9 contiguous amino acids from antigenic site IV,

Any 10 contiguous amino acids from antigenic site IV,

Any 11 contiguous amino acids from antigenic site IV,

Any 12 contiguous amino acids from antigenic site IV.

Any 5-20 contiguous amino acids from antigenic site I, presented above;

Any 5-15 contiguous amino acids from antigenic site I,

Any 5-12 contiguous amino acids from antigenic site I,

Any 5-10 contiguous amino acids from antigenic site I,

Any 6-11 contiguous amino acids from antigenic site I,

Any 6-10 contiguous amino acids from antigenic site I,

Any 7-10 contiguous amino acids from antigenic site I,

Any 8-10 contiguous amino acids from antigenic site I,

Any 6 contiguous amino acids from antigenic site I,

Any 7 contiguous amino acids from antigenic site I,

Any 8 contiguous amino acids from antigenic site I,

Any 9 contiguous amino acids from antigenic site I,

Any 10 contiguous amino acids from antigenic site I,

Any 11 contiguous amino acids from antigenic site I,

Any 12 contiguous amino acids from antigenic site I.

The following specific sequences are used in VLPs according to thepresent invention:

From Peptide F (See FIG. 1 hereof):

S10.1 (SEQ ID NO: 8) LTNSELLSLI S10.2 (SEQ ID NO: 9) SELLSLINDM S10.3(SEQ ID NO: 10) LSLINDMPIT S10.4 (SEQ ID NO: 11) INDMPITNDQ S10.5(SEQ ID NO: 12) MPITNDQKKL S10.6 (SEQ ID NO: 13) TNDQKKLMSN S10.7(SEQ ID NO: 14) DQKKLMSNNVFrom Peptide G (See FIG. 1 hereof):

G16 (SEQ ID NO: 15) PCSICSNNPTCWAICK G14 (SEQ ID NO: 16) CSICSNNPTCWAICG12 (SEQ ID NO: 17) SICSNNPTCWAI G7 (SEQ ID NO: 18) CSNNPTC G5(SEQ ID NO: 19) SNNPT

Any one or more of the above epitopic peptides can be incorporated intoVLPs according to the present invention in order to produce a vaccineagainst RSV infection or a RSV-related disorder.

Production of Virus-Like Particles

The present invention is directed to virus-like phage particles as wellas methods for producing these particles in vivo or in vitro. Themethods typically include producing virus-like particles (VLPs) andrecovering the VLPs. As used herein, producing VLPs “in vitro” refers toproducing VLPs outside of a cell, for instance, in a cell-free system,while producing virions “in vivo” refers to producing VLPs inside acell, for instance, an Eschericia coli or Pseudomonas aeruginosa cell.

Bacteriophages

The single-strand RNA bacteriophages are a group of viruses found widelydistributed in nature that infect diverse bacteria. These bacteriophagescontain a single-stranded (+)-sense RNA genome, contain maturase, coatand replicase genes, and have small (<300 angstrom) icosahedral capsids.Members of this family include, but are not limited to, MS2, PP7, Qβ,R17, SP, PP7, GA, M11, MX1, f4, Cb5, Cb12r, Cb23r, 7s and f2 RNAbacteriophages.

Several phage in this family have been characterized in great detail interms of genome sequence, molecular biology, and capsid structure andassembly. MS2 is perhaps the best studied member of the group. MS2 has a3569-nucleotide single-strand RNA genome that encodes only fourproteins: maturase, coat, lysis and replicase. The viral particle iscomprised of 180 coat polypeptides, one molecule of maturase, and onecopy of the RNA genome. In contrast to many other bacteriophages, theRNA phages are surprisingly simple. In fact, because the coat proteinitself is responsible for formation of the icosahedral shell, the VLPsof this family of bacteriophage can be produced from plasmids as theproduct of a single gene (28). Likewise, PP7, a single-strand RNAbacteriophage of Pseudomonas aeruginosa and a distant relative tocoliphages like MS2 and Qβ, may also be used in the present invention.

Examples of PP7 coat polypeptides include but are not limited to thevarious chains of PP7 Coat Protein Dimer in Complex With RNA Hairpin(e.g. Genbank Accession Nos. 2QUXR; 2QUXO; 2QUX_L; 2QUX_I; 2QUX_F; and2QUX_C). See also Example 1 herein and (22). Examples of MS2 coatpolypeptides include but are not limited to the Crystal Structure of MS2coat protein (e.g. Genbank Accession Nos. 1MSCA; 1ZDIC; 1ZDIA; 1ZDIB;and 6MSFA). Other coat polypeptides are also useful in the presentinvention, as set forth hereinabove.

PP7 and MS2 Coat Polypeptides

The coat polypeptides useful in the present invention also include thosehaving similarity with one or more of the coat polypeptide sequencesdisclosed above. The similarity is referred to as structural similarity.Structural similarity may be determined by aligning the residues of thetwo amino acid sequences (i.e., a candidate amino acid sequence and theamino acid sequence) to optimize the number of identical amino acidsalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. A candidate amino acidsequence can be isolated from a single stranded RNA virus, or can beproduced using recombinant techniques, or chemically or enzymaticallysynthesized. Preferably, two amino acid sequences are compared using theBESTFIT algorithm in the GCG package (version 10.2, Madison, Wis.), orthe Blastp program of the BLAST 2 search algorithm available athttp://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapxdropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, a coatpolypeptide also includes polypeptides with an amino acid sequencehaving at least 80% amino acid identity, at least 85% amino acididentity, at least 90% amino acid identity, or at least 95% amino acididentity to one or more of the amino acid sequences disclosed above.Preferably, a coat polypeptide is active. Whether a coat polypeptide isactive can be determined by evaluating the ability of the polypeptide toform a capsid and package a single stranded RNA molecule. Such anevaluation can be done using an in vivo or in vitro system, and suchmethods are known in the art and routine. Alternatively, a polypeptidemay be considered to be structurally similar if it has similarthree-dimensional structure as the recited coat polypeptide and/orfunctional activity.

The RSV glycoprotein peptide sequences (e.g. the RSV F and Gglycoprotein peptide sequences, preferably the F peptide sequences) maybe present at the amino-terminal end of a coat polypeptide, at thecarboxy-terminal end of a coat polypeptide, or it may be presentelsewhere within the coat polypeptide. Preferably, the RSV glycoproteinpeptide sequences (e.g. the RSV F and G glycoprotein peptide sequences,preferably the F peptide sequences) are present at a location in thecoat polypeptide such that the insert sequence is expressed on the outersurface of the capsid. In a particular embodiment, RSV F and Gglycoprotein peptide sequences (preferably F peptide sequences) may beinserted into the AB loop regions of the above-mentioned coatpolypeptides. Examples of such locations include, for instance,insertion of the insert sequence into a coat polypeptide in accordancewith the examples presented hereinafter.

Alternatively, the RSV glycoprotein peptide sequences (e.g. the RSV Fand G glycoprotein peptide sequences, preferably the F peptidesequences) may be inserted at the N-terminus or C-terminus of the coatpolypeptide.

The RSV glycoprotein peptide sequences (e.g. the RSV F and Gglycoprotein peptide sequences, preferably the F peptide sequences)include but are not limited to amino acid sequences of, at least, three,five, ten, fifteen, twenty or thirty amino acids derived from the RSV Fand G glycoprotein peptide sequences of RSV Types A and B.

In another particular embodiment, the RSV glycoprotein peptide sequenceepitopes includes amino acid sequences with at least 75%, 80%, 85%, 90%or 95% homology to sequences derived from RSV Types A and B.

In order to determine a corresponding position in a structurally similarcoat polypeptide, the amino acid sequence of this structurally similarcoat polypeptide is aligned with the sequence of the named coatpolypeptide as specified above.

In a particular embodiment, the coat polypeptide is a single-chain dimercontaining an upstream and downstream subunit. Each subunit contains afunctional coat polypeptide sequence. The RSV glycoprotein peptidesequences (e.g. the RSV Fund G glycoprotein peptide sequences,preferably F peptide sequences) may be inserted in the upstream and/ordownstream subunit at the sites mentioned herein above, e.g., AB loopregion of downstream subunit. In a particular embodiment, the coatpolypeptide is a single chain dimer of a PP7 coat polypeptide.

Preparation of Transcription Unit

The transcription unit of the present invention comprises an expressionregulatory region, (e.g., a promoter), a sequence encoding a coatpolypeptide and transcription terminator. The RNA polynucleotide mayoptionally include a coat recognition site (also referred to a“packaging signal”, “translational operator sequence”, “coat recognitionsite”). Alternatively, the transcription unit may be free of thetranslational operator sequence. The promoter, coding region,transcription terminator, and, when present, the coat recognition site,are generally operably linked. “Operably linked” or “operably associatedwith” refer to a juxtaposition wherein the components so described arein a relationship permitting them to function in their intended manner.A regulatory sequence is “operably linked” to, or “operably associatedwith”, a coding region when it is joined in such a way that expressionof the coding region is achieved under conditions compatible with theregulatory sequence. The coat recognition site, when present, may be atany location within the RNA polynucleotide provided it functions in theintended manner.

The invention is not limited by the use of any particular promoter, anda wide variety of promoters are known. The promoter used in theinvention can be a constitutive or an inducible promoter. Preferredpromoters are able to drive high levels of RNA encoded by the codingregion encoding the coat polypeptide Examples of such promoters areknown in the art and include, for instance, the lac promoter, T7, T3,and SP6 promoters, among others.

The nucleotide sequences of the coding regions encoding coatpolypeptides described herein are readily determined. These classes ofnucleotide sequences are large but finite, and the nucleotide sequenceof each member of the class can be readily determined by one skilled inthe art by reference to the standard genetic code. Furthermore, thecoding sequence of an RNA bacteriophage single chain coat polypeptidecomprises a site for insertion of RSV glycoprotein peptide (e.g. the RSVF and G glycoprotein peptide and as otherwise described herein)-encodingsequences. In a particular embodiment, the site for insertion of the RSVglycoprotein peptide (e.g. the RSV F and G glycoproteinpeptide)-encoding sequence is a restriction enzyme site.

In a particular embodiment, the coding region encodes a single-chaindimer of the coat polypeptide. In a most particular embodiment, thecoding region encodes a modified single chain coat polypeptide dimer,where the modification comprises an insertion of a coding sequence atleast four amino acids at the insertion site. The transcription unit maycontain a bacterial promoter, such as a lac promoter or it may contain abacteriophage promoter, such as a T7 promoter and optionally a T7transcription terminator.

In addition to containing a promoter and a coding region encoding afusion polypeptide, the RNA polynucleotide typically includes atranscription terminator, and optionally, a coat recognition site. Acoat recognition site is a nucleotide sequence that forms a hairpin whenpresent as RNA. This is also referred to in the art as a translationaloperator, a packaging signal, and an RNA binding site. Without intendingto be limiting, this structure is believed to act as the binding siterecognized by the translational repressor (e.g., the coat polypeptide),and initiate RNA packaging. The nucleotide sequences of coat recognitionsites are known in the art. Other coat recognition sequences have beencharacterized in the single stranded RNA bacteriophages R17, GA, Qβ, SP,and PP7, and are readily available to the skilled person. Essentiallyany transcriptional terminator can be used in the RNA polynucleotide,provided it functions with the promoter. Transcriptional terminators areknown to the skilled person, readily available, and routinely used.

Synthesis

As will be described in further detail below, the VLPs of the presentinvention may be produced in vivo by introducing transcription unitsinto bacteria, especially if transcription units contain a bacterialpromoter Alternatively VLPs synthesized in vitro in a coupled cell-freetranscription/translation system.

Assembly of VLPs Encapsidating Heterologous Substances

As noted above, the VLPs of the present invention display a RSVglycoprotein peptide (e.g. the RSV F and G glycoprotein peptide)-encoding sequence. These VLPs may be assembled by performing an invitro VLP assembly reaction. Specifically, purified coat proteinsubunits are obtained from VLPs that have been disaggregated with adenaturant (usually acetic acid). The protein subunits are mixed with aheterologous substance. In a particular embodiment, the substance hassome affinity for the interior of the VLP and is preferably negativelycharged. This substance could include an adjuvant, including, but notlimited to RNA, bacterial DNA (CpG oligonucleotides), cholera toxinsubunit B, or E. coli lymphotoxin,

Synthesis

In a particular embodiment, the populations of the present invention maybe synthesized in a coupled in vitro transcription/translation systemusing procedures known in the art (see e.g. U.S. Pat. No. 7,008,651). Ina particular embodiment, bacteriophage T7 (or a related) RNA polymeraseis used to direct the high-level transcription of genes cloned undercontrol of a T7 promoter in systems optimized to efficiently translatethe large amounts of RNA thus produced.

Uses of VLPs and VLP Populations

There are a number of possible uses for the VLPs and VLP populations ofthe present invention. As will be described in further detail below, theVLPs may be used to as immunogenic compositions, particularly vaccines.

Immunogenic compositions

As noted above, the VLPs of the present invention may be used toformulate immunogenic compositions, particularly vaccines. The vaccinesshould be in a form that is capable of being administered to an animal.Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat a condition or disorder. Upon introduction into ahost, the vaccine is able to provoke an immune response including, butnot limited to, the production of antibodies and/or cytokines and/or theactivation of cytotoxic T cells, antigen presenting cells, helper Tcells, B cells, dendritic cells and/or other cellular responses.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention. The term “adjuvant”as used herein refers to non-specific stimulators of the immune responseor substances that allow generation of a depot in the host which whencombined with the vaccine of the present invention provide for an evenmore enhanced immune response. A variety of adjuvants can be used.Examples include complete and incomplete Freund's adjuvant, aluminumhydroxide, and modified muramyl dipeptide.

Optionally, the vaccine of the present invention additionally includesan adjuvant which can be present in either a minor or major proportionrelative to the compound of the present invention.

EXAMPLES

The invention may be better understood by reference to the followingnon-limiting examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.References corresponding to numerical reference citations are listedafter the examples.

Materials and Methods. 1. Plasmids.

Previously we described the construction of the expression plasmidspDSP62 and p2P7K32 (6). U.S. Provisional Patent Application Ser. Nos.61/335,120, filed Dec. 31, 2009, 61/302,836, filed Feb. 9, 2010,61/334,826, filed May 14, 2010, PCT application no. PCT/US2010/62638,filed 31 Dec. 2010 and PCT application no. PCT/US2011/24030, filed 8Feb. 2011, all of which applications are incorporated by referenceherein in their entirety. These plasmids code for the expression of aversion of MS2 (pDSP1 or pDSP62) or PP7 (p2P7K32 or pDSP7) coat proteinin which two copies of coat protein are genetically fused into a“single-chain” dimer. These plasmids also contain unique restrictionsites that allow for genetic insertion of sequences into a region thatencodes the AB-loop of the downstream copy of coat.

Details regarding these plasmids are presented below.

pDSP1—A Plasmid Expressing a Single-Chain Dimer with Convenient

Cloning Sites for Insertion in the AB-loop.

The plasmid pDSP1 (see FIGS. 3 and 4) contains the T7 transcriptionsignals of pET3d and the kanamycin resistance and replication origin ofpET9d. [Information regarding pET3d and pET9d may be found at the NewEngland Biolabs vector database,https://www.lablife.org/ct?f=v&a=listvecinfo). It expresses the codingsequence of the MS2 single-chain coat protein dimer (29), modified tocontain unique SalI and KpnI restriction sites. This facilitates simplecloning of foreign sequences into the AB-loop. To make these sitesunique, it was necessary to destroy other SalI and KpnI sites in thevector and in the upstream coat sequence.

The MS2 coat sequence (SEQ ID NO: 36) in the vicinity of the AB-loopinsertion site for pDSP1 is shown below. Note the presence of SalI andKpnI sites.

. . . 6  7  8  9  10 11 12 13 14 15 16 17 18 19 20 21 22 . . .. . . GlnPheValLeuValAspAsnGlyGlyThrGlyAspValThrValAlaPro . . .. . . CAGTTCGTTCTCGTCGACAATGGCGGTACCGGCGACGTGACTGTCGCCCA . . .                   SalI        KpnI

With pDSP1, recombinant coat proteins are usually constructed by cloninginto the AB-loop a PCR fragment generated using a monomeric coat proteinsequence as template (e.g. pMCT). A synthetic oligonucleotide 5′-primeris designed to attach a SalI (or KpnI) site and a sequence of codons(corresponding to the insert sequence) to a site just upstream of theAB-loop (as shown in FIG. 1). A 3′-primer anneals to sequences in theplasmid vector just downstream of BamHI. The resulting PCR product isdigested with SalI (or KpnI) and BamHI and cloned at the correspondingsites of pDSP1. This results in insertion of peptides into the AB-loop,the exact site of insertion depending on the specific design of the5′-primer. For most insertions use of the SalI site is preferred as itaffords more flexibility that KpnI in selection of the insertion site.FIG. 5 shows the nucleotide sequence of the plasmid pDSP62, which issimilar to pDSP1 in that it serves as a vector for expression of asingle-chain dimer form of the MS2 coat protein. In FIG. 5 the coatprotein sequence has been modified so that a peptide derived from RSV Fantigen (peptide S10.5, see FIG. 1) has been inserted into thedownstream copy of coat protein at the AB-loop. A similar plasmid(pDSP7) has been designed to express a single-chain dimer version of thePP7 coat protein. This plasmid also allows for facile insertion ofheterologous sequences into the PP7 AB-loop.

The Plasmid p2P7K32.

It should be noted that the three-dimensional structure of the PP7capsid shows that it is comprised of a coat protein whose tertiarystructure closely mimics that of MS2, even though the amino acidsequences of the two proteins show only about 12% sequence identity(33). The PP7 protein possesses an AB-loop into which peptides may beinserted following a scheme similar to the one we described previouslyfor MS2 (30).

FIG. 6 depicts the p2P7K32 plasmid. This plasmid contains the lacpromoter, an ampicillin resistance cassette, and the ColE1 replicationorigin. It expresses the coding sequence of the PP7 single-chain coatprotein dimer modified to contain a unique KpnI restriction site only inthe downstream copy of the coding sequence (5, 6). This modificationresulted in the amino acid substitution E11T. In this design,heterologous peptides can be inserted at amino acid 11, but it should benoted that other specific insertion sites are possible, possiblyanywhere within the AB-loop.

Recombinant MS2 and PP7 VLPs Displaying RSV Epitopes.

MS2 and PP7 coat protein single-chain dimers are highly tolerant ofpeptide insertions and produce correctly assembled VLPs displaying thepeptide insertion on the surface of VLP in a highly dense, repetitivearray. These VLPs are highly immunogenic and confer this highimmunogenicity to heterologous peptides displayed on their surfaces.Here, we show the ability to make recombinant VLPs displaying RSVepitopes and show that these VLPs are highly immunogenic.

EXAMPLE 1

Here we describe VLPs displaying a peptide antigens derived from RSV Fand G glycoprotein peptides. To create the VLPs that display RSVpeptides we designed oligonucleotide primers that allowed us to clonevarious RSV F- and G-derived sequences by PCR or primer extension intothe AB-loop of either MS2 or PP7 coat. The predicted amino acidsequences of selected constructs are shown in FIG. 1. RSV sequences wereselected as described below:

Targeting RSV F Protein.

A region of the RSV F glycoprotein mapping roughly to amino acids252-272 has been identified as a major neutralizing epitope (23). BothMS2 and PP7 coat protein dimers can readily accept 10 amino acidinsertions. Thus, we created a series of F protein chimeras in whichlinear 10 amino acid sequences were inserted into the downstream copy ofthe MS2 and PP7 single-chain dimers (shown in FIG. 1).

Targeting RSV G Protein.

Based on analysis of escape mutants, a region of RSV G glycoproteinbetween amino acids 164 and 197 is thought to be a major neutralizingepitope (24). Part of this region is fairly hydrophobic (aa 164-174) andit includes a pair of disulfide bonds forming a loop (between cysteines173 and 186 and between cysteines 176 and 182). The region from aa181-197 has a C3xC motif, the putative viral receptor binding region,has been used to generate antibodies, and is not hydrophobic incharacter. Thus, we have designed a series of constructs within thisregion that contain the cysteine loop plus flanking sequences or justthe cysteine loop itself, as shown in FIG. 1.

The ability of recombinant coat proteins displaying RSV F and Gsequences to form VLPs in lysates of cells expressing a peptide-coatprotein recombinant was tested by electrophoresis on agarose gel ofcells lysed by sonication. Ethidium bromide staining detects theRNA-containing VLP. Table 1 below shows the ability of recombinant MS2and PP7 coat proteins to form VLPs. As shown, the vast majority of F andG recombinants resulted in VLPs.

TABLE 1 Ability of recombinant MS2 and PP7 coat proteins to form VLPsInsertion MS2 PP7 G5 +++^(a) +++ G7 +++ +++ G12 + ++ G14 − + G16 − +10.1 + nt^(b) 10.2 + nt 10.3 ++ nt 10.4 +++ nt 10.5 +++ nt 10.6 +++ nt10.7 +++ nt ^(a)+++ high yield of VLPs, ++ medium yield, + low yield, −no VLPs ^(b)nt, Not Tested

EXAMPLE 2

RSV Peptides Displayed on MS2 VLPs Induce Antibodies that Bind to RSVVirions.

To test the immunogenicity of the VLPs, mice were immunized withselected recombinant MS2 VLPs by intramuscular injection. Groups ofthree Balb/c mice were immunized intramuscularly with 10 μg of VLPs plusincomplete Freunds Adjuvant (IFA). All mice were boosted with the sameamount of VLPs at weeks 2 and 6. Sera were collected before eachinoculation and weekly for three to four weeks after the boost. Serafrom the mice were tested, by ELISA, for IgG antibodies specific for apeptide representing amino acids 252-272 from F protein or recombinant Fprotein, or for IgG antibodies specific for inactivated RSV virions(FIG. 2). As shown in FIGS. 2a and 2b , all of the F protein recombinantVLPs induced high-titer antibodies that bound to the synthetic F peptideand recombinant F protein. Three of the recombinant F VLPs (10.5, 10.6,& 10.7; FIG. 2c ) and one of the recombinant G VLPs (G5; FIG. 2d )showed reactivity with RSV virions.

Summary of Experimental Results.

Genetic display of peptides on PP7 VLPs is well suited for the precisetargeting of specific B-cell epitopes known to be the target ofneutralizing antibodies. For many pathogens, including influenza (15,32), Hepatitis C Virus (20), and HIV (4), the target epitopes of broadlyneutralizing antibodies are poorly immunogenic, meaning that full-lengthproteins are inadequate for the induction of antibody responses byvaccination. On the other hand, the use of peptide epitopes as vaccinesis limited because of their poor immunogenicity unless coupled tocarrier proteins. The PP7 and MS2 VLP platforms that we have describedallow for targeted introduction of specific peptide epitopes in a highlyimmunogenic context. Here, we show that MS2 and PP7 recombinant coatprotein displaying the neutralizing epitopes derived from RSV G and Fglycoproteins can form VLPs. VLPs displaying certain F and G epitopesinduce antibodies that can bind to RSV virions. These recombinant VLPscan serve as a prophylactic vaccine for RSV infection.

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1-60. (canceled)
 61. An immunogenic RNA bacteriophage virus-likeparticle (VLP) comprising an RNA bacteriophage single chain coatpolypeptide dimer comprising (i) an upstream coat protein subunit, (ii)a downstream coat protein subunit, and (iii) a heterologous RespiratorySyncytial Virus (RSV) glycoprotein F peptide that consists of an aminoacid sequence corresponding to 5 to 15 contiguous amino acids of SEQ IDNO:5.
 62. The RNA bacteriophage VLP of claim 61, wherein that VLP is aPP7 or MS2 VLP.
 63. The RNA bacteriophage VLP of claim 61, wherein theRSV peptide is inserted at the amino terminus of the single chain dimerpolypeptide.
 64. The RNA bacteriophage VLP of claim 61, wherein the RSVpeptide is inserted in the AB loop of the downstream subunit.